Distinct Interactions Select and Maintain a Specific Cell Fate Andreas Doncic, Melody Falleur-Fettig, Jan M. Skotheim  Molecular Cell  Volume 43, Issue 4, Pages 528-539 (August 2011) DOI: 10.1016/j.molcel.2011.06.025 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 43, 528-539DOI: (10.1016/j.molcel.2011.06.025) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Start Marks the Point of Commitment to the Cell Cycle, where Cells Lose Mating Competence (A and B) Schematics of G1 cell cycle and pheromone-induced MAPK pathway regulation (mutual inhibition indicated with red arrows). (C) Whi5-GFP enters the nucleus right before cytokinesis and is exported from the nucleus in G1 and is a quantitative marker of cell cycle progression. A histone-mCherry fusion protein marks the nucleus. (D and E) Composite phase and green fluorescence images showing cells growing in a microfluidic device exposed to changing α-factor concentrations shown in (E). (F) Time course of the nuclear Whi5-GFP from the two example cells in (D) marked with red and blue outlines. Molecular Cell 2011 43, 528-539DOI: (10.1016/j.molcel.2011.06.025) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Nuclear Whi5-GFP Accurately Predicts Cell Fate (A and B) Time courses of nuclear Whi5-GFP for a pre-Start (A) and a post-Start (B) cell at the time of pheromone addition (dashed line). For each cell we record the fraction of Whi5-GFP exported from the nucleus prior to α-factor exposure (δ/γ), the time spent in G1, cell size, type, and subsequent cell fate: arrest (A) or cell cycle commitment (B). (C) Histograms and logistic regression curve (inset) based on cell fate (red, arrest; blue, commit) as determined by the fraction of exported Whi5 at the time of pheromone addition. The shaded region in the logistic curve indicates a 95% confidence intervals based on 10,000 bootstrapping iterations. (D–F) Corresponding histograms based on cell area (D), and time spent in G1 (E), cell type (F). (G) Cell fate predictions for specified models. Molecular Cell 2011 43, 528-539DOI: (10.1016/j.molcel.2011.06.025) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Induction of the CLN2 Promoter Correlates with Exporting Half the Whi5-GFP from the Nucleus (A) CLN2pr expression (top) and the nuclear Whi5-GFP concentration (bottom) were monitored in single cells. Each color corresponds to a specific cell. For each cell we note relative amount of Whi5-GFP (δ/γ as in Figure 2) at the time of CLN2pr induction (solid line). Measurements were performed in cln1Δcln2Δ cells (see text). (B) Histogram of the exported Whi5-GFP fraction at the time of CLN2pr induction. The amount of Whi5 exported by the time of CLN2pr induction was 0.49% ± 1% (mean ± SEM) and is similar to the amount exported prior to Start in WT cells (52% ± 3%). Molecular Cell 2011 43, 528-539DOI: (10.1016/j.molcel.2011.06.025) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Genetic Analysis of Start Reveals the Primacy of the Cln1/2-Far1 Interaction in Determining Commitment to Cell Division (A–F) For each genotype, we performed the Whi5-based quantitative Start assay as shown in Figure 2C for WT cells. Cells were grown for 2 hr prior to exposure to a step increase in pheromone concentration. Subsequent cell fate (arrest directly or commit to an additional division) and nuclear Whi5-GFP concentration were measured. Each panel shows a histogram of the nuclear Whi5-GFP concentration (δ/γ as shown in Figures 2A and 2B) at the time of pheromone addition for each cell fate (red, arrest; blue, commit). For each mutant, we used logistic regression to estimate the probability of arrest as a function of nuclear Whi5-GFP, which is always compared to the corresponding curve for WT cells shown in black. The shaded regions indicate 95% confidence intervals estimated from 10,000 bootstrapping iterations. Indicated p values are computed relative to WT using a χ2 test. The number of cells used for each mutant is NWT = 315, NSTE5-8A = 218, NFAR1-S87A = 273, Ncln1Δcln2Δ = 477, Ncln3Δ = 266, NFAR1-S87A STE5-8A = 216, N3XFAR1 = 193. (G) Summary of the genetic analysis reveals two clusters (WT, STE5-8A, cln3Δ) and (FAR1-S87A, cln1Δcln2Δ, FAR1-S87A STE5-8A, 3XFAR1). Each mutant is not significantly different from the other mutants in its cluster (p > 0.07 for all comparisons) but significantly different from all mutants in the other cluster (p < 0.002 for all comparisons; see Table S2). Molecular Cell 2011 43, 528-539DOI: (10.1016/j.molcel.2011.06.025) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Ste5 Phosphorylation Is Required for Cell Fate Exclusivity (A and B) Composite phase and fluorescence images of WT (A) and STE5-8A (B) cells exposed to α-factor in late G1. The wild-type cell buds (pink arrow) and shmoos normally (white arrow), whereas a fraction of the STE5-8A cells first bud (pink arrow) and progress through a partial, or as seen here more complete, anaphase before fusing the two nascent nuclei and shmooing (white arrow). (C) τ is the time between cell cycle commitment, defined as half the Whi5-GFP peak nuclear concentration, and α-factor addition. (D) Error frequency as a function of τ reveals that STE5-8A cells just after the commitment point often mix cell fates. All wild-type, all far1Δ, and STE5-8A cells exposed to α-factor pre-Start do not arrest aberrantly. Since STE5-8A far1Δ cells always attempt a cell cycle, Whi5-GFP can exit the nucleus after α-factor addition (τ < 0). (E and F) Example single-cell gene expression from integrated FUS1pr-GFP and CLN2pr-mCherry gene expression reporters in WT (E) and STE5-8A (F) cells. (G) Mean (± SEM) coexpression of CLN2 and FUS1-reporters. Coexpression for single cells was obtained by multiplying background subtracted and peak-scaled green and red signals. STE5-8A cells were separated into cells with aberrant arrest, prolonged mitosis, or normal arrest. Aberrant arrest and prolonged mitosis phenotypes correlate significantly with mixed gene expression. Molecular Cell 2011 43, 528-539DOI: (10.1016/j.molcel.2011.06.025) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 A Separation of Kinetic Timescales May Result in a Separation of Function (A) Schematic of the simplified model (see text and Supplemental Information for details). We analyze the effects of STE5-8A and FAR1-S87A mutations by setting k1 or k3 to zero, respectively. (B) The relative impact of the STE5-8A and FAR1-S87A mutations as a function of the ratio between their inhibition rates. The commitment point is set by the fastest timescale: if k1 > > k3, Ste5 inhibition primarily determines commitment (right-hand side of B); however, if k1 < < k3, Far1 inhibition primarily determines commitment (left-hand side of B). (C) Schematic of the experimental protocol and fluorescence images of an example cell expressing STE5-YFP and FAR1-Venus. Upon α-factor arrest, Far1 accumulates in the nucleus and Ste5 on the shmoo tip. Next, cells exposed to media containing pheromone but lacking methionine to induce CLN2 expression from an integrated MET3 promoter. We measure the initiation of Far1 degradation and Ste5 membrane dissociation. (D) Cumulative distribution for the time at which Far1 (blue curve) and Ste5 (red curve) inhibition is detected. Molecular Cell 2011 43, 528-539DOI: (10.1016/j.molcel.2011.06.025) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Cell Cycle Commitment at Start Is Determined by CLN1/2-Positive Feedback in Competition with Far1 Ste5 inhibition is required to truncate the pheromone signal postcommitment to ensure the exclusive expression of the mitotic transcriptional program. Molecular Cell 2011 43, 528-539DOI: (10.1016/j.molcel.2011.06.025) Copyright © 2011 Elsevier Inc. Terms and Conditions